PROJECT SUMMARY This proposal is based on a newly discovered regulatory arm of the cellular stress response, whereby Jun N- terminal kinase (JNK) is recruited to translating ribosomes (polysomes) to mediate degradation of newly synthesized proteins (NSPs). We established the significance of this novel regulatory module to breast cancer (BCa) biology, and identify ubiquitin proteasome system (UPS) components that?for the first time?are linked with the surveillance of NSPs. They include Cullin 2, Nedd8, and ubiquilin1 (UBQLN1), which we demonstrate to impact protein synthesis in BCa cells. Dysregulated expression of these UPS components in BCa underlies the rationale for studying their role in BCa development and response to therapy. Our preliminary results support the hypothesis that control of NSP stability constitutes a novel layer of regulation of protein synthesis/availability, which in turn governs cellular responses to stress. We further hypothesize that such regulation has direct implications for BCa development and response to therapy. We focus on several complementary but hitherto unappreciated mechanisms that may underlie NSP surveillance under stress. The productive and long-standing collaborations between Drs. Topisirovic, Sonenberg, Mills and Ronai are now extended to include Dr. Meller, thereby enabling extensive and complementary expertise in the areas of protein synthesis and cancer biology to also include nanopore-sensing technology, enabling the resolution of ubiquitin chain topologies. Together, we will assess specific, newly identified NSP regulatory factors that function individually or in concert to regulate the cellular stress response, particularly in the context of BCa development and response to therapy. The proposed research will: (1) Establish the physiological significance of the RACK1?JNK?eEF1A2 regulatory axis to the cellular stress response, growth and therapeutic response of breast cancer. (2) Assess the role of stress-induced polysomal recruitment of Nedd8?Cullin machinery in regulating the decay of NSPs in BCa. (3) Determine the importance of UBQLN1 recruitment to polysomes in regulating newly synthesized proteins under stress conditions and in modulating the response of BCa to therapy. Our proposed studies will establish the importance and significance of select UPS components in a novel regulatory network that controls protein synthesis during cellular stress, and establish its role in BCa using a combination of BCa cultures and xenografts, RPPA technology, and TCGA dataset mining.